WO2010128156A1 - Dérivés de 2,1,3-benzoxadiazole pour l'inhibition de la réplication du virus de la grippe a et b et du virus respiratoire syncytial - Google Patents

Dérivés de 2,1,3-benzoxadiazole pour l'inhibition de la réplication du virus de la grippe a et b et du virus respiratoire syncytial Download PDF

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WO2010128156A1
WO2010128156A1 PCT/EP2010/056294 EP2010056294W WO2010128156A1 WO 2010128156 A1 WO2010128156 A1 WO 2010128156A1 EP 2010056294 W EP2010056294 W EP 2010056294W WO 2010128156 A1 WO2010128156 A1 WO 2010128156A1
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nitro
benzoxadiazole
thio
benzoxadiazol
influenza
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PCT/EP2010/056294
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English (en)
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Ulrich Kessler
Charlene Ranadheera
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Pike Pharma Gmbh
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Priority to EP10719756A priority Critical patent/EP2427442A1/fr
Priority to US13/318,763 priority patent/US20120122896A1/en
Publication of WO2010128156A1 publication Critical patent/WO2010128156A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D271/00—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms
    • C07D271/12—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms condensed with carbocyclic rings or ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4245—Oxadiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/167—Purine radicals with ribosyl as the saccharide radical

Definitions

  • the present invention relates to small molecules inhibiting the replication of influenza A and B virus and respiratory syncytial virus (RSV), and the use of such compounds for treating influenza A and B and RSV infections, in humans, mammals and birds.
  • RSV respiratory syncytial virus
  • Influenza viruses are negative-stranded RNA viruses that cause yearly epidemics as well as recurring pandemics, resulting in high numbers of human cases and severe economic burden.
  • pandemic influenza A viruses such as the 1918 "Spanish" flu or H5N1
  • pandemic influenza B viruses contribute greatly to the annual recurring epidemics that cause the vast majority of human cases and medical cost.
  • the WHO recommends an annual vaccination against circulating influenza A (FIuA) and B (FIuB) strains.
  • FIuA circulating influenza A
  • FIuB circulating influenza A
  • current vaccines confer incomplete protection against epidemic influenza.
  • oseltamivir oseltamivir
  • RelenzaTM zanamivir
  • RSV Human respiratory syncytial virus
  • SSV Human respiratory syncytial virus
  • RNA virus RNA virus of the family Para myxovi rid a e
  • Treatment is mainly limited to supportive care, including oxygen.
  • Palivizumab (SynagisTM) is used as a prophylactic drug in prevention of respiratory RSV infections for infants with a high risk of infection.
  • Ribavirin has been used for treating RSV infections, but showed limited effectiveness.
  • One object of the invention is to provide new, improved and/or alternative influenza and RSV antiviral compounds.
  • Another object of the invention is to obviate or mitigate disadvantages of influenza antiviral agents and RSV antiviral agents known from the state of the art.
  • a compound as a medicament a compound for treating influenza type A and/or influenza type B and/or RSV infections in humans, mammals and/or birds, the use of a compound for the manufacture of a medicament for the treatment of influenza type A and/or in- fluenza type B and/or RSV infections in humans, mammals and/or birds, and a pharmaceutical composition comprising such a compound, according to the independent claims.
  • Advantageous embodiments are given in the dependent claims.
  • the 2,1 ,3-benzoxadiazole compounds as a medicament according to the invention are: 4-[(4-methoxybenzyl)thio]-7-nitro-2,l ,3-benzoxadiazole,
  • the above defined compounds according to the invention are particular advantageous for treating and/or preventing influenza type A and/or influenza type B infections in humans, mammals and/or birds; as well as for treating and/or preventing respiratory syncytial virus (RSV) infections in humans, mammals and/or birds.
  • RSV respiratory syncytial virus
  • the compound according to the invention can be used for the manufacture of a medicament for the treatment and/or prevention of influenza type A and/or influenza type B infections in humans, mammals and/or birds, and/or for the treatment and/or prevention of respiratory syncytial virus infections in humans, mammals and/or birds.
  • a pharmaceutical composition according to the invention comprises a compound according to the invention.
  • Advantageously such a composition comprises one or more excipients.
  • compounds in accordance with the present invention are able to inhibit protein-protein interaction of the PA and PBl subunits of the heterotrimeric viral RNA polymerase complex of both influenza virus types A and B, and thus are able to inhibit replication of influenza A and B virus.
  • the viral polymerase subunit interaction domain turned out as an effective target for the new antiviral compounds, since correct assembly of the three viral polymerase subunits PBl , PB2 and PA is required for viral RNA synthesis and infectivity. Structural data for the entire trimeric complex is missing.
  • the crucial PA interaction domain of PBl consists of a 3io-helix formed by amino acids (amino acids 5-1 1 ).
  • the domain is highly conserved and virus type specific among both, influenza A and B viruses.
  • an Enzyme-Linked Immunosorbent Assay (ELISA) based screening assay and other assays are used to prescreen compounds according to the invention that show antiviral activity against influenza A and B viruses. Since they are effective against both virus types, such compounds represent an attractive alternative to neuraminidase inhibitors. Therefore, the present invention represents a major step toward a sorely needed, near-universal medicament against influenza virus, and one which, due to its protein- protein interaction domain target, will likely be less susceptible to the emergence of drug-resistant strains for which influenza is well known.
  • RSV respiratory syncytial virus
  • the compounds according to the invention can be used as a medicament, particularly as an influ- enza virus and/or RSV replication inhibitor and an influenza and/or RSV preventive/therapeutic agent, respectively.
  • Influenza Type A and B Therapeutic target
  • ammo acids are preferably indicated by the IUPAC one letter code in the present application. Whenever three letter codes are used, they are also in accordance with IUPAC.
  • the letter X is used to indicate a wildcard/variable or other ammo acid at a certain position.
  • Table I a shows the inhibitory concentrations of FluA/FluB-derived peptides determined by competitive ELISA. Competitor peptides (0.048 to 300OnM) were mixed with cell extracts containing HA-tagged PA from either FIuA or FIuB. Table 1 lists 1 2 competitive peptides. The first peptide PBI i 15 A is the FIuA wild type, the second row shows the FIuB wild type. For the peptides of rows 3 to 8 letters indicate FIuB specific ammo acids. Rows 9 to 1 2 list further competitive peptides with ammo acids at position 6 being neither FIuA nor FIuB specific. Standard deviation is indicated in parenthesis.
  • Asterisks indicate highest concentrations of peptides used without reaching 50% inhibition.
  • Further competitive peptides which are not listed in the table but have effectively reached 50% inhibition at low peptide concentrations are PBl i-i 5 A T6 i , PBl i-i 5 A T6L and PB 1 1 _i 5 A T6 v ⁇ Peptides with slightly lower inhibition activity are PB 1 H 5 A T 6 A and PBl i-i 5 A T6M which are also not shown in Table I a.
  • Table I a inhibitory concentrations of FluA/FluB-derived peptides determined by competitive ELISA
  • the synthesized or isolated influenza virus replication-inhibiting peptides interacting with the inhibition target for the small molecules compounds according to the invention comprise an ammo acid sequence of X 5 X 6 X 7 XsXgXi O , wherein X 5 is P; X 6 is T, Y, F, W, H, C, I, L, V, A or M; X 7 is L or F; X 8 is L, I, F or M; X 9 is 5 F, Y, W, H, L, R or S, and Xi 0 is L, I or Y.
  • Said ammo acid sequence is at least 60 %, preferably at least 70%, more preferably at least 80% or 90% identical to the polypeptide according to the wild type PBI i 1 1 A which is M DVN PTLLFLK.
  • those peptides are preferred which comprising the ammo acid sequence of X 6 X 7 X 8 XgXi O , wherein X 6 is T, Y, F, W, H, C, I, L or V; X 7 is L or F; X 8 is L or I; Xg is F, Y or W and Xi 0 is L
  • Even more preferred according to certain embodi- i o ments are peptides that comprise the ammo acid sequence of X 6 X 7 , wherein X 6 is T, Y, F, W, H, C, I, L or V and X 7 is L or F.
  • Effective peptides advantageously comprise at least 1 1 residues Xm , whereby preferably the proteins comprise the ammo acid sequence M DVN PX6X7 LFLKVPAQ wherein X6 is selected from the group: T, Y, F, W, H. C, A, I, L, V or M and X7 is selected from the group L or F.
  • a preferred peptide comprises an 15 ammo acid sequence elected from the group: MDVNPYFLFLKVPAQ, MDVNPYLLFLKVPAQ, MDVNPWLLFLKVPAQ or MDVNPFLLFLKVPAQ.
  • the peptides comprise at least 1 5 residues Xi ] 5 according to the wild type PBl i ] 5 A but not the wild type sequence MDVN PTLLFLKVPAQ.
  • Table 2 shows the 50%- ⁇ nh ⁇ b ⁇ tory concentrations (IC 5 o) of FluA-derived PBl peptides determined by 20 competitive ELISA.
  • Peptide PBI i 25 A was immobilized on microwell plates and incubated with increasing concentrations of competitor peptides and cell extract containing HA-tagged PA of FIuA. Bound PA was detected by HA-specific antibodies as described above. Standard deviation is shown in parenthesis. Asterisks indicate highest concentrations of peptides used without detectable inhibitory effect.
  • Grey boxes highlight ammo acids that are part of the 3i o-hel ⁇ x, which comprises the core PA-binding region of PBl . 25 Ammo acids known to form hydrogen bonds with PA residues are represented in bold.
  • Table 3 illustrates the inhibitory concentrations (IC 50 ) of FluA-derived competitor peptides determined by ELISA. Peptide PBI 1 25 A was again immobilized on microwell plates and incubated with increasing concentrations of competitor peptide and cell extract containing HA-tagged PA of FIuA. HA-specific antibodies detected bound PA. Standard deviations are shown in parenthesis. Asterisks indicate highest concentrations of peptides used without detectable inhibitory effect. Table 3: Inhibitory concentrations (IC 50 ) of FluA-derived PBl peptides
  • Figure 1 shows binding and inhibitory activity of PB11-25AT6Y. Based on Figure 1 the binding and inhibitory activity of peptides binding to the inhibition target with a focus on the preferred protein PBl]. 25 A T6 ⁇ shall be illustrated in the following part of the description.
  • Figure Ia shows in the upper panel the alignment of the consensus sequence of the N-terminal 25 amino acids of FIuA and FIuB PBl, wherein the dotted box indicates the 3io-helix comprising the core PA-binding domain of PBl and the FIuA- specific and FluB-specific amino acids are printed in bold letters.
  • Middle and lower panels show the alignment of the N-terminal 25 amino acids of all available FIuA and FIuB sequences derived from PBl full length sequences provided by the NCBI influenza virus database.
  • the binding of HA-tagged PA subunits from cell extracts to the immobilized peptides corresponding to the domains of FIuA PBl (PBl 1 25 A), FIuB PBl (PBl 1 25 B) or FIuA PBl T6Y (PBl 1 25 A ⁇ 6 ⁇ ) determined by ELISA is shown in Figure I b. Signals using the cognate peptide and lysate were normalized to 1 . Binding of the PA subunits to the control peptides was not observed.
  • Upper panels Western blot of the PA- 5 containing cell extracts used. Molecular weights shown in kilodaltons.
  • Figure I c provides graphic information on the structure of FIuA PBl 1 1 5 bound to FIuA PA.
  • T6 forms a hydrogen bond to a water molecule.
  • Molecular modeling suggests that the aromatic side chain in the mutant T6Y fits into a hydrophobic pocket and displaces the water molecule.
  • the polymerase inhibitory activity of PBl 1 25 -der ⁇ ved CFP fusion proteins in FIuA and FIuB polymerase reconstitution assays is i o shown in Figure I d.
  • the activity in experiments containing all viral plasmids and Flag-CFP was set to 1 00%.
  • Figure I e shows a plaque reduction assay using PB1 1 25 A-Tat; PBl 1 25 A T6 ⁇ -Tat; PX-Tat (control peptide) with FIuA, FIuB and VSV (vesicular stomatitis virus).
  • a H 2 O control was used to standardize the assay to 1 00%.
  • PB1 1 25 B-Tat could not be tested due to insolubility. Error bars represent standard de- 15 viations.
  • Figure 2a shows A/SC35M- and B/Yamagata/73-der ⁇ ved PBl chimeras used in tests according to Figure 2b. Note that all PBl proteins were expressed with C-terminal HA-tags.
  • Figure 2b shows human 293T cells which were transfected with expression plasmids coding for the indicated PBl proteins and the C-terminally hexahistidme-tagged PA
  • FIuA 20 of FIuA (FIUAPAH IS ).
  • IP immunopre- cipitation
  • aHA ant ⁇ -HA
  • Precipitated material was separated by SDS-PACE and analyzed by Western blot for the presence of either His- or HA-tagged polymerase subunits using appropriate antibodies. Protein expression was controlled by analyzing equal amounts of cell lysate. Molecular weights are shown in kilodaltons.
  • the 25-mer peptide, PBl 1 25 A, comprising a helical domain inhibits
  • FIG 4a shows CFP-PBl fusion proteins used in tests according to Figure 4b.
  • the complex formation of PBl 1 25 -der ⁇ ved CFP fusion proteins and HA-tagged PA of FIuA and FIuB is shown in Figure 4b.
  • Indicated proteins were expressed in human 293T cells and binding of the CFP fusion proteins was analyzed by immunoprecitation (IP) of PA using ant ⁇ -HA agarose and subsequent immunoblotting (IB). Precipitated material was analyzed by Western blot using the indicated antibodies for the presence of either HA- tagged PA or CFP.
  • IP immunoprecitation
  • IB immunoblotting
  • Virus strains For the infection experiments A/WSN/33 (Hl N l ) according to Chanem et al. (2007) and A/Tha ⁇ land/l (Kan-l )/2004 according to Chockephaibulkit et al. (2005), B/Yamagat/73 according to Norton (1987) and VSV (serotype Indiana) as described in Schwemmle (1995) were used.
  • Plasmid constructions Plasmids pCA-Flag-CFP and pCA-PBl 1 25 A-CFP, pCA-PBl -HA, the FIuA minireph- con plasmids and the expression plasmids for the FIuB minireplicon are described in Chanem (2007), Mayer (2007) and Pleschka (1996).
  • the FIuB minigenome expression plasmid, pPoll-lucRT_B was ob- tamed by cloning the firefly luciferase ORF (inverse orientation) flanked by the non-coding region of the segment 8 of the B/Yamagata/73 into the Sapl-digested plasmid pPoll-Sapl-Rib according to Pleschka (1996).
  • pCA-PBl 1 25 B-CFP For the construction of pCA-PBl 1 25 B-CFP, a linker containing the first 25 codons of PBl (B/Yamagata/73) was cloned into the EcoRI/Notl sites of pCA-Flag-CFP plasmid, replacing the Flag- coding sequence with PBI 1 25 B. Site directed mutagenesis was carried out with pCA-PBl 1 25 A-CFP to create the plasmid pCA-PBl 1 25 A T6 rGFP.
  • the ORFs of PBl (B/Yamagata/73) and PA (A/SC35M, A/Tha ⁇ land/1 (KAN-I )/04, A/V ⁇ etnam/1 203/04, B/Yamagata/73, B/Lee/40) were PCR amplified with sense primers containing an Notl site (FIuA strains) or a EcoRI site (FIuB strains) upstream of the initiation codon and antisense primers with a deleted stop codon followed by an Xmal site, a coding sequence for an HA-tag and a Xhol site.
  • PCR products were cloned into a modified pCACCsvector (Schneider, 2003) digested either with EcoRI/Xhol or Notl/Xhol, resulting in pCA-PBl -HA or pCA-PA- HA plasmids, coding for C-terminal tagged versions of the polymerase subunits.
  • pCA- PA / yscss M -His plasmid pCA-PA / yscss M -HA was digested with Xmal/Xhol and the HA coding sequence was replaced by a 6xHis-l inker.
  • the A/B-chimeric expression plasmids were obtained by assembly PCR using the pCAPBl -HA plasmids of SC35M and B/Yamagata/73 and by cloning the resulting PCR product in pCA-PBl B / ⁇ amagata / 73 -HA digested with EcoRI/EcoRV.
  • HEK293T cells were transiently transfected with a plasmid mixture containing either FIuA- or FluB-derived PBl-, PB2-, PA- and NP-expression plasmids, polymerase I (Pol l)-driven plasmid transcribing an influenza A or influenza B virus-like RNA coding for the reporter protein firefly luciferase to monitor viral polymerase activity and with expression plasmids coding for the indicated CFP fusion proteins. Both minigenome RNAs were flanked by non-coding sequences of segment 8 of FIuA and FIuB, respectively.
  • the transfection mixture also contained a plas- mid constitutively expressing Renilla luciferase, which served to normalize variation in transfection efficiency.
  • the reporter activity was determined 24h post transfection and normalized using the Dual-Clu® Lufierase Assay System (Promega). The activity observed with transfection reactions containing Flag-CFP were set to 100%.
  • Peptide synthesis The solid-phase synthesis of the peptides was carried out on a Pioneer automatic peptide synthesizer (Applied Biosystems, Foster City, USA) employing Fmoc chemistry with TBTU/diisopropylethyl amine activation. Side chain protections were as follows: Asp, CIu, Ser, Thr and Tyr: t-Bu; Asn, CIn and His: Trt; Arg: Pbf; Lys and Trp: Boc. Coupling time was 1 h. Double couplings were carried out if a difficult coupling was expected according to the program Peptide Companion (Coshi- Soft/PeptiSearch, Arlington, USA).
  • pep- tides After precipitation with t-butylmethylether, the resulting crude pep- tides were purified by preparative HPLC (RP-18) with water/acetonitrile gradients containing 0.1 % TFA and characterized by analytical HPLC and MALDI-MS. Some peptides were synthesized by pep- tides&elephants (Nuthetal, Germany) and subsequently purified and characterized as described above.
  • HEK293T cells were transfected with the indicated plasmids in 6- well plates using Metafectene (Biontex, Martinsried, Germany). Cells were incubated 24h post transfec- tion with lysis buffer (2OmM Tris pH7.5, 10OmM NaCI, 0.5mM EDTA, 0.5% NP-40, 1 % Protease inhibitor Mix G, (Serva, Heidelberg, Germany), I mM DTT) for 1 5 min on ice. After centrifugation by 1 3.000 rpm at 4°C supernatant was incubated with anti HA-specific antibodies coupled to agarose beads (Sigma) for 1 h at 4°C.
  • lysis buffer 2OmM Tris pH7.5, 10OmM NaCI, 0.5mM EDTA, 0.5% NP-40, 1 % Protease inhibitor Mix G, (Serva, Heidelberg, Germany), I mM DTT
  • Plaque reduction assay The experiments were carried out as described by Schmidke (2001 ) with modi- fications. Confluent MDCK cells were infected with I OOPFU of A/WSN/33, B/Yamagata/73, A/KAN-] , or VSV/lndiana in PBS containing BSA at room temperature. After removal of the inoculum, cells were overlaid with medium (DMEM with 2OmM Hepes, 0.01 % DEAE Dextran, 0.001 % NaHC03) containing 1 % Oxoidagar and candidate peptides or small molecule compounds at the indicated concentrations.
  • medium DMEM with 2OmM Hepes, 0.01 % DEAE Dextran, 0.001 % NaHC03
  • Enzyme-Linked Immunosorbent Assay For the ELISA microwell plates (Pierce) were incubated with saturating concentrations of peptides at room temperature, washed and subsequently incubated at room temperature with HA-tagged PA. To obtain PA-HA, 293T cells were seeded into 94mm-dishes, transfected with the respective plasmid and treated with lysis buffer 24h post transfection as described in detail by Mayer et al. (2007).
  • HA-specific primary antibody Covance
  • a peroxidase- coupled secondary antibody Jackson lmmuno Research, Newmarket, UK
  • the competition ELISA was carried out as described above with the exception that the candidate peptide or small molecule competitor compound were added to wells of the plate with bound peptides prior to addition of the cell extract containing HA-tagged PA subunits.
  • the test sample includes a known binding pair of proteins or protein subunits including a fluorescent label, which can be analyzed according to a preferred embodiment of the present invention by fluorescence polarization.
  • a fluorescent label which can be analyzed according to a preferred embodiment of the present invention by fluorescence polarization.
  • PBl Influenza A virus polymerase subunit
  • PA subunit PA
  • the test sample is then contacted with a candidate peptide or small molecule inhibitor compound and the resulting fluorescence polarization is determined.
  • the ability of the compound to cause dissociation of or otherwise interfere with or prevent binding of the proteins or protein subunits is monitored by fluorescence polarization (FP).
  • FP measurements allow for discrimination between fluorescently labeled bound and unbound proteins, peptides, subunits or fragments thereof.
  • the FP of the fluorescently Ia- beled first fragment rotates rapidly in solution and, therefore, has randomized photo-selected distributions, which result in the small observed FP.
  • the rotation of the fluorescently labeled first fragment slows and the fluorescence polarization increases. Accordingly, disruption of the subunit interaction by a test compound provides a de- crease in the fluorescence polarization, which is indicative of inhibition of the protein interactions.
  • the FP measurements in the presence of a test compound can be compared with the FP measurements in the absence of the test compound. Comparison can be made manually by the operator or automatically by a computer, especially in high throughput assays using 384-well plates.
  • PA protein purification influenza
  • a virus polymerase subunit PA was cloned into a suitable expression vector with a C-terminally attached 6xHis-linker or hemagglutinine epitope (HA).
  • Human 293T cells were transfected with the plasmid.
  • Cell lysates were prepared 24 hours post transfection using lysis buffer (2OmM TrisHCI pH 7.5, 10OmM NaCI, 0.5mM EDTA, 0.5% NP-40, I mM DTT and 1 % Protase inhibitor mix)
  • PA subunit was bound to Ni- or anti-HA-agarose and washed with lysis buffer without protease mix.
  • PA-protein was concentrated when necessary using Vivaspin20 5OK columns and frozen at -8O 0 C until further use. After thawing, the elution buffer was exchanged to low fluorescent grade reagents and any HA-peptide was removed simultaneously using 10-DC Bio-Cel columns.
  • Fluorescently labeled peptide corresponding to the 25 first N-terminal amino acids of Influenza A virus polymerase subunit PBl at 3 nM concentration was added to l O ⁇ M HA-PA in 2OmM TrisHCI pH 7.5, 1 5OmM NaCI, 0.5mM EDTA, I mM DTT, 5% Glycerol and l OOmg/ml bovine gamma globulin. The mix was distributed into black 384-well plates to a total volume of 20 ⁇ l per well and kept on ice. Test com- pounds solved in DMSO were added to a final concentration of 25 ⁇ M. After incubation for 10 minutes at room temperature, plates were read using an Infinite F200 reader (Tecan). FP values of the wells containing test compounds were compared to wells without test compounds, without DMSO and with peptide only.
  • Sequence alignment Alignments were performed with MUSCLE as described in Edgar (2004) using the full-length sequences provided from the public influenza virus database (http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html).
  • HEp-2 cells obtained from ATCC were seeded in 96-well plates (1 .5 x 104 cells per well) and grown in MEM-alpha medium containing 10% FBS (Gibco-BRL) for 24 h.
  • MEM-alpha medium containing 10% FBS (Gibco-BRL) for 24 h.
  • OptiMEM OptiMEM
  • Protein-protein interactions are crucial to most, if not all, biological processes. Of the roughly 30,000 protein sequences that comprise the human proteome, only about 1 % have been successfully targeted with small-molecule drugs. Yet, most of the conventional targets in drug discovery fall into the same few structural or functional families such as enzymes or C protein-coupled receptors (CPCRs). They typically share the property that the natural substrates or ligands, with which they interact are themselves small organic molecules. Historically there has been notably little success in developing drug-like inhibitors of proteins whose natural ligands are other proteins.
  • the present invention uses the fact that proteomes of many viruses and PPIs crucial for viral replication are described in the literature. For any proteome of interest, this data is according to the novel method supplemented with proteomic approaches for identification of PPIs like yeast two-hybrid or co-immuno precipitation screening in order to identify potential target regions for development of PPI inhibitors. Subsequently, a unique combination of phylogenetic analysis and structure prediction or structure analysis (where applicable) of the protein partners involved detects druggable protein-binding domains.
  • druggable denotes preferably protein-binding domains which can be blocked, altered or modified by small molecules in a way that the protein-protein interaction is inhibited or disrupted.
  • small molecules denotes organic molecules, preferably synthetic organic molecules (not peptides), which have a molecular weight below 1 500, preferably below 1000 and most preferred below 500 u. It has been found, that these domains bear a couple of characteristic features: (i) helical structure, (ii) hydrophobic character and (iii) high conservation among all virus strains. It has been shown that they tend to be located at a terminal end of the protein or are located on their surface. The peptides corresponding to these potential binding domains are synthesized in an overlapping way and tested for their ability to bind the protein partner involved in the PPI.
  • peptides resembling short, (less than 20 amino acids) continuous binding domains are identified, these are used for the development of a binding assay, preferably an ELISA or fluorescence polarization (FP) assay, which is afterwards employed in a high-throughput screening campaign for small molecule and/or peptidic inhibitors of the PPI.
  • a binding assay preferably an ELISA or fluorescence polarization (FP) assay
  • PPI inhibitors identified by the novel method according to the present invention could offer a particular advantage when it comes to antivirals since it should be safe to assume that resistance development occurs at a much slower pace.
  • PKE060 4-[(4-methoxybenzyl)th ⁇ o]-7-n ⁇ tro-2,l ,3-benzoxad ⁇ azole M / KM06831 Yes
  • PKE075 4-[(2,4-d ⁇ chlorophenyl)th ⁇ o]-7-n ⁇ tro-2,l ,3-benzoxad ⁇ azole M / KM06828 Yes
  • PKE080 4-[(3-chlorophenyl)th ⁇ o]-7-n ⁇ tro-2,l ,3-benzoxad ⁇ azole M / KM06838 Yes
  • PKE081 2-[(7-n ⁇ tro-2,l ,3-benzoxad ⁇ azol-4-yl)th ⁇ o]ethyl 2,4- M / KM06863 No dichlorobenzoate
  • PKE082 2-[(7-n ⁇ tro-2,l,3-benzoxad ⁇ azol-4-yl)th ⁇ o]ethyl-4 M/KM06867 Yes -methoxybenzoate
  • PKEl 33 4-n ⁇ tro-l-ox ⁇ do-7-[4-(phenylmethyl)p ⁇ peraz ⁇ n-l-yl]-2,l,3- N/NSC228099 No benzoxad ⁇ azol-1- ⁇ um
  • PKEl 38 2-[2-am ⁇ no-6-[(4-n ⁇ tro-2,l,3-benzoxad ⁇ azol-7-yl) sulfanyljpu ⁇ n- N/NSC348400 Yes 9-yl]-5-(hydroxymethyl)oxolane-3,4-d ⁇ ol
  • the inhibitory concentrations (IC 5 o) have been determined (Table 5), in a plaque reduction assay as described above for the influenza peptide studies or with a competitive ELISA assay as described above for the influenza peptide studies. In cases where the solubility was too low to reach the saturation region, the IC 5O value was calculated based on the inhibition on the maximum obtainable concentration. If an IC 5O value was not obtained, maximum ELISA inhibition at the highest concentration used (1000 ⁇ fvl) is given. Table 5: Influenza inhibitory concentrations (IC 5 o) of compounds
  • the compounds that have been found so far to be effective in binding to PA have a basic structure of 2,1 ,3,-benzoxadiazole.
  • compound PKE060 has an IC 5O that is considerably lower than PKE079, PKE080, PKE082, PKEl 07, PKEl 08, PKEl 37, and PKEl 38.
  • the IC 50 of the other compounds is not sufficiently low to be physiologically acceptable.
  • the assessed class of 2,1 ,3,-benzoxadiazole based compounds seems to be effective in the inhibition of replication of certain virus types.
  • a number of compounds effectively inhibited the replication of influenza virus, particularly influenza A namely compounds PKE 060, PKE 079, PKE 080, PKE 082, PKE 107, PKE 108, PKE 137, and PKE 138.
  • the compounds according to the invention can be very effective broad band inhibitors of virus replication, and thus are a valuable source of effective new me- dicaments against certain types of the orthomyxoviridae and paramyxoviridae families.

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Abstract

Un composé de 2,1,3-benzoxadiazole en tant que médicament selon la présente invention est l'un des composés suivants : le 4-[(4-méthoxybenzyle) thio]-7-nitro-2,1,3-benzoxadiazole, le 4-méthoxybenzène-1-sulfonate de 2-[(7-nitro-2,1,3-benzoxadiazol-4-yle) thio] éthyle, le 4-[(4-méthylphényle) thio]-7-nitro-2,1,3-benzoxadiazole, le 4-[(2,4-dichlorophényle) thio]-7-nitro-2,1,3-benzoxadiazole, le 2-[(7-nitro-2,1,3-benzoxadiazol-4-yle) thio] éthan-1-ol, le 4-[(4-méthylbenzyle) thio]-7-nitro-2,1,3-benzoxadiazole, le 4-[(4-fluorophényle) thio]-7-nitro-2,1,3-benzoxadiazole, le 4-[(3-chlorophényle) thio]-7-nitro-2,1,3-benzoxadiazole, le 4-méthoxy-benzoate de 2-[(7-nitro-2,1,3-benzoxadiazol-4-yle) thio] éthyle, le 5-[4-(tert-butyle)-1,3-thiazol-2-yle]-2,1,3-benzoxadiazole, la N-benzyl-4-nitro-2,1,3-benzoxadiazol-5-amine, le 4-nitro-7-(phénylméthylsulfanyle)-2,1,3-benzoxadiazole, le 4-nitro-7-(phénylméthylsulfonyle)-2,1,3-benzoxadiazole, le 2-(hydroxyméthyle)-5-[6-[(4-nitro-2,1,3-benzoxadiazol-7-yle) sulfanyle] purin-9-yle] oxolane-3,4-diol, ou le 2-[2-amino-6-[(4-nitro-2,1,3-benzoxadiazol-7-yle) sulfanyle] purin-9-yle]-5-(hydroxyméthyle) oxolane-3,4-diol; ou un sel, un solvate physiologiquement acceptable, ou un dérivé physiologiquement fonctionnel de ceux-ci. Lesdits composés sont particulièrement avantageux pour le traitement et/ou la prévention des infections dues à la grippe de type A et/ou à la grippe de type B chez les humains, les mammifères et/ou les oiseaux, et pour le traitement et/ou la prévention des infections dues au virus respiratoire syncytial chez les humains, les mammifères et/ou les oiseaux.
PCT/EP2010/056294 2009-05-08 2010-05-07 Dérivés de 2,1,3-benzoxadiazole pour l'inhibition de la réplication du virus de la grippe a et b et du virus respiratoire syncytial WO2010128156A1 (fr)

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